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Zahedan Journal of Research in Medical Sciences Antibacterial Effect of Myrtus Communis Hydro-Alcoholic Extract on Pathogenic Bacteria Ali Taheri,*1 Amir Seyfan,2 Samira Jalalinezhad,3 Fatemeh Nasery4
1. Department of Fisheries (Seafood Sciences), Faculty of Marine Sciences, Chabahar Maritime and Marine Sciences University, Chabahar, Iran 2. Department of Fisheries, Faculty of Marine Sciences, Chabahar Maritime and Marine Sciences University, Chabahar, Iran 3. Medical Student, Zahedan Medicine University, Chabahar International Branch (Sina), Sistan and Balouchestan, Iran 4. Department of Agricultural Biotechnology, Iranian Veterinary Organization, Chabahar Office, Sistan and Balouchestan, Iran
Article informationAbstract Background: Today, due to the changes in the form of the resistance of pathogenic
bacteria, discovering new antimicrobial drugs is under study. So, the aim of this study is to
evaluate the antimicrobial properties of the extract of the myrtle herb on some of
Materials and Methods: Hydroalcoholic extract of the leaves of myrtle herb was
evaluated at 4 concentrations including 10-80 mg/ml on four strains of pathogenic bacteria
using penetrative dissemination method together with the measuring diameter of the
growth inhibition zone; then the results were compared to four conventional antibiotics.
The minimum inhibitory and bactericidal concentrations were studied using macro dilution method.
Results: Treatment by the concentration of 80 mg/ml extract of this herb showed the
Department of Fisheries (Seafood Sciences), Faculty of
greatest effect on the bacterium Staphylococcus aureus and Vibrio cholera serotype
Ogawa which had a significant difference with all other treatments and standard antibiotics
(p< 0.05). The extract showed no effect on the bacterium Pseudomonas aeruginosa and
just concentration of 80 mg/ml showed a little effect on E. coli and other antibiotics had no
significant effect except tetracycline which has little effect on this strain. Minimum
inhibitory concentration was 0.2 mg/ml for bacterium Staphylococcus aureus (S. aureus) and the maximum for E.coli by 8 mg/ml. Conclusion: This study showed that under study bacteria were more resistant to the antibiotics and the extract of Myrtus communis leaves showed greatest antibacterial effect against S. aureus and V. cholerae cerotype Ogawa.
Copyright 2013 Zahedan University of Medical Sciences. All rights reserved.
Introduction
oday, environmental problems and high prices of
Khorasan River Valley, Sarab, Guilan-e-gharb, Kerman,
some antibiotics have reinforced a tendency to
Maharlu in Shiraz, Neyriz, Fasa, Mamasani and Bandar
replace them with less harmful substances. Among
Abbas [5]. The dried leaves of this herb contain
the various materials to replace antibiotics, products of
terpineolene, cineol, linalool, terpineole, linalyl acetate,
plant origin have recently gotten a special place [1, 2].
tannins and flavonoids compounds and there are
Herbal medicines have been the only source of pain
numerous reports about the anti-parasitic and anti-
treatment during the centuries and now with the
infective properties of the extract of this herb [6- 10].
advancement of sciences and development in the
Also its anti-virus effect has led to the production of anti-
application of synthetic drugs, medicinal plants are still
used in large-scale [3]. This issue is very important for the
Today, the anti-parasitic and antimicrobial drug
application in drug therapy or side applications such as
resistance is a major problem in the world which is due to
the uncontrolled use of antimicrobial drugs. This
However, due to the climatic variation and the vast area
resistance is so important that the theme of 2011 World
of Iran, wide spectrum of medicinal plants are found there
Health Organization was "Resistance to antimicrobial
which are the basis of Iran's traditional medicine [4-6].
drugs is a global threat". This resistance can be
Thus, in recent years, extensive researches have been
transmitted by microbes from one generation to another
conducted to evaluate the antimicrobial effect of the
generation and even from a microbial species to another
essential oils and extracts which shows the strength and
species through the creation of an antibiotic-resistant gene
ability of these compounds to inhibit the growth of a wide
by them and ultimately the high levels of infection
range of pathogenic microorganisms [7]. Myrtle (Myrtus
remained stable despite the administration of antibiotics.
communis) of the Mytaseae family is a shrub, evergreen
Until recently, several studies have been conducted on the
and aromatic herb with numerous stems and branches.
antimicrobial properties of the leaf and stem extracts of
This herb is found in the white Lab of Bakhtiari valley,
myrtle against pathogenic bacteria and good results have
Zahedan J Res Med Sci 2013 June; 15(6): 19-24.
been obtained about its effects on staphylococcus aureus
Concentration of bacteria became equal to the
[12-4], E. coli [15-16], Lactobacillus plantarum [16],
McFarland standard tube No. 0.5 (108×1.5) after 24 h
Bacillus cereus [12], Listeria monocytogenes and
incubation in the logarithmic phase of growth, using a
Pseudomonas aeruginosa, [17-18], Klebsiella and
spectrophotometer. This suspension was considered as a
Shigella [17], though its antimicrobial effect against some
reserve and diluted in a similar medium based on 1:100
strains of bacteria such as E. coli has been rejected in
ratio while taking on the same day (6 10×5/1) [23].
Susceptibility to the nosocomial microorganisms to the
Some gram-negative bacteria such as E. coli and
myrtle extract was conducted using penetrative
Pseudomonas aeruginosa and some gram-positive
dissemination method. Bacterial suspension was seeped
bacteria such as Staphylococcus aureus have specific
on the medium using 20 ml sampler and was spread on
characteristics as far as bacterial infections are concerned
the medium with a sterile cotton swap; then it was dried
and they are removed or isolated from the most of the
for 5 minutes and incubated for 15 minutes in order to
clinical samples referred to the diagnostic laboratories
equilibrate the moisture inside the plates.
[19, 20]. On the other hand, some reports on the cases of
Sterile crude discs with 6 mm diameter were placed on
being infected with cholera by Vibrio cholerae which is a
the surface of medium and 15 microliters of the myrtle
gram-negative bacterium are annually published and
solution was seeped in a specified concentration on the
being infected with this bacterium has been observed in
discs. Standard discs of streptomycin 10, gentamicin 10
the southern districts of the country due to the
and erythromycin 15 and tetracycline 30, produced by
consumption of contaminated raw or semi raw fishes and
vegetables irrigated with sewage. Accordingly, given the
Plates were incubated for 24 hours at 37°C and bacterial
importance of medicinal plants in the traditional medicine
inhibition zone was measured and recorded using Vernier
and very few side effects of these medicines on human
calipers. Experiments were conducted with three
and also due to the changes in the form of the resistance
replications. The minimum growth inhibitory
of pathogenic bacteria which requires monitoring of
concentration and Minimum Lethal Concentration (MLC)
antibacterial effects substances in periodic intervals, the
of antimicrobials were determined using the tube dilution
antimicrobial effects of hydroalcoholic extract of Myrtus
method [24, 25]. A series of nine test tubes were used for
communis leaves against the above-mentioned pathogenic
each concentration and each bacterium. There were 7
bacteria have been evaluated in this study.
tubes for different dilutions, a positive control tube and a
negative control tube. 9 ml nutrient broth was added to
Materials and Methods
the test tubes and then was sterilized. 1000 microliters of
the diluted extract was added to the first tube and after
This study was conducted in 2011 at the Laboratory of
being homogenized, 1000 microliters of the homogenized
Microbiology of Chabahar University of Maritime and
fluid was added to the second tube and this operation was
Marine Sciences. Extraction was carried out with slight
continued and 1000 microliters of homogenized solution
variations based on the study conducted by Sadeghi [21].
(from 7th tube) was discarded. 10 microliters of the
Accordingly, the leaves of myrtle were collected in spring
bacterial suspension was added to all tubes except the
from Zaranjan located in the district of Fasa and they
negative control tube based on McFarland tube 1.
were dried in the shade after washing. 600 grams of the
All tubes were incubated for 24 h at 37 C and then the
ground leaf powder was mixed with 500 ml of distilled
tubes were evaluated in terms of turbidity due to the
water and ethyl alcohol 96 at 1:1 ratio and was kept in a
bacterial growth. The last tube in which no turbidity was
dark place for 48 hours. The contents of the Erlenmeyer
seen was considered as the minimum growth inhibitory
flasks were stirred for 25 minutes once every 16 hours.
concentration. The solutions inside all tubes with no
After 48 hours, the contents of the Erlenmeyer flask were
growth turbidity were cultured using pour plate method
smoothed by the filter paper and smoothed fluid was
for determining minimum bactericidal concentrations of
extracted in the rotary evaporation apparatus under
the myrtle extract and the final concentration of the
vacuum and 50̊C temperature. The concentrated extract
extract which was able to kill 99.9% of the live bacteria,
was poured in sterile Petri dish and dried in the oven with
40̊C. Dried powders were collected and were prepared at
concentration of the microorganisms [26, 27].
concentrations of 10, 20, 40 and 80 mg using sterile
Statistical analysis: Experiments were performed in
distilled water. Staphylococcus aureus, Pseudomonas
three replications and Graphpad-Prism 7.00 software was
aeruginosa, Vibrio cholerae serotype Ogawa and E. coli
used for the data analysis under analysis of variance test;
Bacteria were prepared from the samples of patients
and Duncan test was used to compare treatments at 95%
referred to Imam Ali hospital of Chabahar city after doing
specialized microbiologic tests for confirming the
presence of microorganisms. Cultures were purred and
stored at refrigeration temperature until the start of the
study [22]. One day before experiments, a small portion
Table 1 shows the results of penetrating emissions test
of the mother culture was added to the Hinton broth
of the myrtle extract with different concentrations on
under-study microorganisms. Accordingly, the
concentration of 80 mg/ml of myrtle extract showed the
Antibacterial effect of myrtus communis Taheri A et al.
greatest effect on the Staphylococcus aureus bacterium
Table 2. Comparison of the minimum growth inhibitory concentration
and no significant difference was seen with all other
and minimum bactericidal concentrations of the myrtle leaf extract on microorganisms
treatments and control antibiotics and showed a higher
efficacy (p<0.05). Lower concentrations also showed a
significant difference with each other and all of them had
significant differences with antibiotics except the
concentration of 10 mg/ml that had no significant
difference with gentamicin (p<0.05). Erythromycin and
streptomycin did not show any effect on bacteria. Myrtle
extract, at any concentration, did not show any effect on
the bacterium Pseudomonas aeruginosa and the highest
Discussion
effect of control group of antibiotics was observed in
gentamicin which had a significant difference with
In the present study, hydroalcoholic extract of the myrtle
leaves showed the greatest effect on Staphylococcus
The study conducted on the effects of myrtle extract on
aureus which had significant difference with the standard
E. coli bacteria also showed that only the concentration of
antibiotics. The positive effect of the myrtle extract on
80 mg/ml has the effect on this strain of bacteria and
Vibrio cholerae serotype Ogawa was well. In this study
except for the very light effect of tetracycline on this
on these two strains of bacteria, concentration of the used
strain, other control antibiotics did not have any effect.
extract also caused a significant difference in the results.
Concentration of 80 mg/ml showed the highest effect on
Myrtle extract showed a very little effect on E. coli only
the Vibrio chlorea Bacterium which had a significant
at the highest concentration but Pseudomonas aeruginosa,
difference with all other treatments (p<0.05).
was completely resistant to the extract.
Lower concentrations also showed a positive effect, all
For centuries in Iran, several herbs have been used to
of them had a significant difference with each other
treat diseases. Different generations' trust in the treatment
(p<0.05). Tetracycline was the only antibiotic effective on
with the traditional medicine demonstrates the positive
this bacteria which had a significant difference with other
impact of this type of treatment. On the other hand, the
concentrations except that concentrations of 10 and 20
increasing resistance of pathogenic bacteria and
mg/ml (p<0.05). Results of minimum inhibitory
continuous change of the resistance form of these
concentration of growth and minimum bactericidal
microorganisms have led to the major challenges in the
concentrations after 24 h culture were shown in table 2.
use of common antibiotic drugs which requires the search
According to the results of this table, myrtle extract had
for the new compounds with anti-bacterial properties.
no effect on the bacterium Pseudomonas aeruginosa.
Myrtle is an aromatic and medicinal herb for which
Minimum inhibitory concentration belonged to the
antibacterial, antifungal, antiviral, antioxidant and anti-
bacterium Staphylococcus aureus and the highest rate
mutagenicity properties have been reported [28-32].
belonged to the E. coli. Similar results were obtained
Positive properties of the myrtle extract on hospital
about the minimum bactericidal concentration.
bacteria have been reported. In a study, effects of several
plants, including myrtle against Streptococcus Table 1. Comparison of the inhibition zone diameter (mm) of myrtle pneumoniae, Haemophilus influenzae and Moraxella
extracts and standard antibiotics on under study microorganisms
catarrhalis isolated from the patients in hospitals were
evaluated and the results showed that the myrtle extract
creates 30, 50 and 22 mm growth inhibition zone in the
diffusion penetration test; therefore the use of myrtle
extract for the treatment of sinusitis and bronchitis was
The positive effect of the myrtle extract on S. aureus in
this study was consistent with the results obtained by
Salvagnini. These researchers were studied the effect of
the oil and ethanolic extract of myrtle on several strains of
bacteria and reported that the ethanolic extract of myrtle
has a positive effect on S. aureus with 12 mm inhibition
zone. But the oil has shown a greater effect [29]. Alem
also reported the antibacterial effects of the myrtle extract
on S.aureus isolated from human samples with 0.5 mg/ml
of the minimum inhibitory concentration [14].
Ghlamhsynyan Najjar et al. who had observed a very
positive effect of the chloroform, ethyl acetate and
* There was no inhibition zone; treatment of any columns indicated
methanol extracts of the myrtle leaves on S. aureus
significant difference with dissimilar letters
acknowledged that antimicrobial effects of myrtle extract
on this bacterial strain is partly related to the stimulation
of free radicals and the concentration of the extract has an
Zahedan J Res Med Sci 2013 June; 15(6): 19-24.
effect on antibacterial activity [34]. In the study of control
the bacteria [36- 38]. Due to this reason, a gram-positive
bacterium is more susceptible to the extract than gram-
concentrations of the myrtle extract on Pseudomonas
No report on the effects of myrtle extract against the
Fewer efficacies of these antibiotics can confirm the
bacteria causing cholera (i.e. Vibrio cholera) has been
resistance of the mentioned strains of bacteria and on the
reported yet. The results of this study show the positive
other hand, various studies have shown the effects of
effect of the hydroalcoholic extract of the myrtle on this
myrtle extract on P. aeruginosa [6, 35].
strain of bacteria; serotype Ogawa which is much better
For example Alem reported the effect of myrtle extract
than that of tetracycline which is the only antibiotic
on Pseudomonas aeruginosa isolated from human cases
effective on this bacterium. In a study conducted by
with a minimum inhibitory concentration of 120 mg/l
Rahbar et al. Vibrio cholerae serotype Inaba and Ogawa
[14]. But the myrtle extract used in this study had no
were sensitive to Tetra Doxycycline, erythromycin and
effect on bacteria which could be due to the mutations in
ampicillin [39]. It can be said that the less efficacy of
the bacterial strains which are still sensitive to antibiotics
antibiotics in this study can be due to the resistance of this
but are resistant to the myrtle extract, although differences
strain of pathogenic bacteria. Several studies have
in the extracted essential oil and even used concentrations
reported the antibacterial activity of essential oil extracted
could be another reason for the lack of conformity with
from the herb. In recent years, many of these researchers
the present findings. In a study conducted by Amensour,
came to this understanding that the presence of phenolic
leaf and stem extract of myrtle was examined on 15
compounds in the leaves of myrtle herb is due to the
strains of bacteria which cause food borne diseases. They
presence of Flavenols (Quercetin glycosides and
reported that the methanol and ethanol extracts of the
myricetin) and derivatives of Galoyl including Galoyl
myrtle leaves and branches have respectively
glycosides, Alajytanyn, the Galvyl–quinic acid [40] and
antimicrobial properties on Listeria monocytogenes,
they attributes the antibacterial activity of these
Pseudomonas aeruginosa and Staphylococcus aureus and
compounds to the presence of polyphenols [41].
the diameters of the inhibition zone of ethanol extract in
Many studies have shown that the mechanism of this
these bacteria are respectively 30, 23 and 37 mm and
effect is due to the influence on the cell wall and reported
minimum bactericidal concentration for Staphylococcus
that the cell wall and cell membrane are affected and their
aureus was reported less than 0.075 mg/ml. Also the
permeability is changed and lead to the release of
highest antibacterial activity was against gram-positive
intracellular contents which can be accompanied with the
bacteria [18]. Comparison of the results of this study
disrupt in the membrane function such as electron
shows that the extract has no effect on Pseudomonas
transfer, enzyme activity or nutrient absorption [18].
aeruginosa and the inhibition zone of Staphylococcus
In sum, it can be said that the results of this study
aureus was about 20 mm in the best condition and the
indicate the antibacterial effect of hydroalcoholic extract
minimum bactericidal concentration for Staphylococcus
of the leaves of myrtle herb on some pathogenic bacteria,
especially Staphylococcus aureus and Vibrio chloreae
The comparison shows the difference between
which can be an alternative for the treatment of diseases
pathogenic bacteria and food born disease agents and may
resulted from these bacterial strains that the resistance
be another proof for the resistance of bacteria studied in
form of which has been changed as an herbal antibiotic
this study. In the studies conducted on the effects of
with side effects fewer than conventional antibiotics. Also
extracts on E. coli, most of the reports show that the
working on other extraction materials and methods such
myrtle extract has no effect on these bacterial strains [18,
as extracting by methanol and ethyl acetate may indicate
29]. In a study conducted in Iran in the past, the positive
different functions. Also clinical confirmation and
effect of alcoholic extract of the myrtle herb on E. coli
pharmacological standardization is required before their
had been reported. Also according to the report of
Ghasemi Pirbalouti et al. although the methanol extract
showed no effect on the diffusion penetrating activity, but
Acknowledgements
the minimum lethal concentration for E. coli was 10
mg/ml [6, 35]. Comparison of the results of this study
We sincerely appreciate the staff of Imam Ali Hospital
revealed that myrtle extract, even in the highest
and Chabahar Veterinary Office for their cooperation with
concentration, has little effect on the diffusion penetration
us. This study has been conducted in the framework of
of E. coli and minimum lethal concentration was
Mr. A. Seyfan's research project of Fisheries Engineering
determined as 40 mg/ml. The resistance of this strain of
at the University of Maritime and Marine Sciences of
pathogenic bacteria could be a reason for this finding. It
Chabahar. Therefore, we appreciate the authorities of this
has been generally reported that the gram-negative
university for the provision of the laboratory facilities for
bacteria are more resistant to the extracts and essential
oils of this herbs, because hydrophilic structure of the cell
walls of the gram-positive bacteria is mainly composed of
Authors’ Contributions
penetration of hydrophobic oil and also prevents
All authors had equal role in design, work, statistical
accumulation of its compounds in the cell membrane of
Antibacterial effect of myrtus communis Taheri A et al.
Conflict of Interest
The authors declare no conflict of interest.
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Please cite this article as: Taheri A, Seyfan A, Jalalinezhad S, Nasery F. Antibacterial effect of Myrtus communis hydro-alcoholic extract on
pathogenic bacteria. Zahedan J Res Med Sci (ZJRMS) 2013; 15(6): 19-24.

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